One of the major impediments facing the development of in vivo applications for monoclonal antibodies in humans is the intrinsic immunogenicity of non-human immunoglobulins. Patients respond to therapeutic doses of rodent monoclonal antibodies by making antibodies against the rodent immunoglobulin sequences. These human anti-mouse antibodies (HAMA) neutralize the therapeutic antibodies and can cause acute toxicity. The MAMA response is less dramatic in immunodeficient patients. Therefore, intrinsic immunogenicity has not prevented the use of rodent monoclonal antibodies for the treatment of graft rejection, which involves the temporary attenuation of the patient's immune response. Rodent antibodies may also be useful for treating certain lymphomas that involve immunodeficiencies. However, even immunodeficient patients can mount a HAMA response which leads to a reduction in safety and efficacy.
The present technology for generating monoclonal antibodies involves pre-exposing, or priming, an animal (usually a rat or mouse) with antigen. This pre-exposure leads to the formation of splenic B-cells that secrete immunoglobulin molecules with high affinity for the antigen. Spleen cells from a primed animal are then fused with myeloma cells to form immortal, antibody secreting, hybridoma cells. Individual hybridoma clones are screened to identify those cells producing immunoglobulins directed against a particular antigen.
The genetic engineering of individual antibody genes has been proposed. Two genetic engineering approaches have been reported: chimeric antibodies and complementarity-determining-region (CDR) grafting. The simplest approach, chimeric antibodies, takes advantage of the fact that the variable and constant portions of an antibody molecule are encoded on separate exons. By simply fusing the variable region exons of a rearranged mouse antibody gene with a human constant region exons, a hybrid antibody gene can be obtained (Morrison, S. L., et al. (1984), Proc. Natl. Acad. Sci. USA, 81, 6851-6855). The major problem with this approach is that while the highly immunogenic mouse Fc region is eliminated, the remaining mouse Fab sequences are still immunogenic (Bruggemann, et al. (1989), J. Exp. Med., 270, 2153-2157). The CDR grafting approach uses computer modeling to generate a completely artificial antibody in which the only mouse sequences are those involved in antigen binding (Riechmann, L., et al. (1988), Nature, 332, 323-327). Each of these approaches requires the prior characterization of a rodent monoclonal antibody directed against the antigen of interest, and both require the generation of a stable transfected cell line that produces high levels of the engineered antibody.
Another approach to the production of human antibodies is a proposal involving the construction of bacterial expression libraries containing immunoglobulin cDNA sequences (Orlandi, et al. (1989), Proc. Natl. Acad. Sci. USA, 86, 3833-3837, and Huse, et al. (1989), Science, 246, 1275-1281). This technique reportedly has only been used to generate antibody fragments derived from mouse cDNA sequences.
A number of experiments have reported the use of transfected cell lines to determine the specific DNA sequences required for Ig gene rearrangement (reviewed by Lewis and Gellert (1989), Cell, 59, 585-588). Such reports have identified putative sequences and concluded that the accessibility of these sequences to the recombinase enzymes used for rearrangement is modulated by transcription (Yancopoulos and Alt (1985), Cell, 40, 271-281). The sequences for V(D)J joining are reportedly a highly conserved, near-palindromic heptamer and a less well conserved AT-rich nanomer separated by a spacer of either 12 or 23 bp (Tonegawa (1983), Nature, 902, 575-581; Hesse, et al. (1989), Genes in Dev., 3, 1053-1061). Efficient recombination reportedly occurs only between sites containing recombination signal sequences with different length spacer regions.
The production of transgenic mice containing various forms of immunoglobulin genes has also been reported. Rearranged mouse immunoglobulin heavy or light chain genes have been used to produce transgenic mice. Such transgenes reportedly are capable of excluding the rearrangement of endogenous Ig genes. See e.g. Weaver et al. (1985), Cell, 42, 117-127; Iglesias, et al. (1987), Nature, 330, 482-484; Storb et al. (1985), Banbury Reports, 20, 197-207; Neuberger et al. (1989), Nature, 338, 350-352; Hagman et al. (1989), J. Exp. Med., 169, 1911-1929; and Storb (1989) in Immunoglobulin Genes, Academic Press, T. Honjo, F. W. Alt and T. H. Rabbitts eds. pp. 303-326. In addition, functionally rearranged human Ig genes including the .mu. or .gamma.1 constant region have been expressed in transgenic mice. Yamamura, et al. (1986), Proc. Nat. Acad. Sci. USA, 83, 2152-2156; Nussenzweig, et al. (1987), Science, 236, 816-819. In the case of the .mu. rearranged heavy chain gene, allelic exclusion of endogenous immunoglobulin gene loci was reported.
Allelic exclusion, however, does not always occur in all transgenic B-cells. See e.g. Rath, et al. (1989), J. Immunol., 143, 2074-2080 (rearranged .mu. gene construct); Manz, et al. (1988), J. Exp. Med., 168, 1363-1381 (.mu. transgenes lacking transmembrane exons did not prevent rearrangement of the endogenous genes); Ritchie, et al. (1984), Nature, 312, 517-520 and Storb, et al. (1986), Immunol. Rev., 89, 85-102 (transgenic mice expressing rearranged .kappa. transgene capable of forming stable heavy/light chain complex only rearrange endogenous .kappa. genes in B-cells that fail to correctly rearrange endogenous heavy chain gene); and Manz, et al. (1988), J. Exp. Med., 168, 1363-1381 (transgenic mice containing .kappa. gene encoding light chain incapable of combining with heavy chains, show only a low level of allelic exclusion). See also Nussenzweig, et al. (1988), Nature, 336, 446-450); Durdik, et al. (1989), Proc. Natl. Acad. Sci. USA, 86, 2346-2350; and Shimizu, et al. (1989), Proc. Natl. Acad. Sci. USA, 86, 8020-8023.
Somatic mutation has also been reported in a 15 kb mouse .kappa. gene construct in hyperimmunized transgenic mice (O'Brien, et al. (1987), Nature, 326, 405-409; Storb (1989) in Immunoglobulin Genes, Academic Press, T. Honjo, F. W. Alt, and T. H. Rabbitts, eds. pp. 303-326) and in the variable portion of a .mu. heavy chain transgene (Durdik, et al. (1989), Proc. Natl. Acad. Sci. USA, 86, 2346-2350).
Ig gene rearrangement, though studied in tissue culture cells, has not been extensively examined in transgenic mice. Only a handful of reports have been published describing rearrangement test constructs introduced into mice [Buchini, et al. (1987), Nature, 326, 409-411 (unrearranged chicken .lambda. transgene); Goodhart, et al. (1987), Proc. Natl. Acad. Sci. USA, 84, 4229-4233) (unrearranged rabbit .lambda. gene); and Bruggemann, et al. (1989), Proc. Natl. Acad. Sci. USA, 86, 6709-6713 (hybrid mouse-human heavy chain)]. The results of such experiments, however, have been variable, in some cases, producing incomplete or minimal rearrangement of the transgene.
Based on the foregoing, it is clear that a need exists for heterologous monoclonal antibodies, e.g. antibodies of human origin, derived from a species other than human. Thus, it is an object of the invention herein to provide a source of monoclonal antibodies that may be used therapeutically in the particular species for which they are designed.
In accordance with the foregoing object transgenic nonhuman animals are provided which are capable of producing a heterologous antibody, such as a human antibody.
Further, it is an object to provide B-cells from such transgenic animals which are capable of expressing heterologous antibodies wherein such B-cells are immortalized to provide a source of a monoclonal antibody specific for a particular antigen.
In accordance with this foregoing object, it is a further object of the invention to provide hybridoma cells that are capable of producing such heterologous monoclonal antibodies.
Still further, it is an object herein to provide heterologous unrearranged and rearranged immunoglobulin heavy and light chain transgenes useful for producing the aforementioned non-human transgenic animals.
Still further, it is an object herein to provide methods to disrupt endogenous immunoglobulin loci in the transgenic animals.
Still further, it is an object herein to provide methods to induce heterologous antibody production in the aforementioned transgenic non-human animal.
A further object of the invention is to provide methods to generate an immunoglobulin variable region gene segment repertoire that is used to construct one or more transgenes of the invention.
The references discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention.